The present invention relates generally to methods for modulating cadherin-mediated processes, and more particularly to the use of modulating agents comprising a cadherin cell adhesion recognition sequence, or an antibody that specifically recognizes such a sequence, for inhibiting or enhancing functions such as cell adhesion.
Cell adhesion is a complex process that is important for maintaining tissue integrity and generating physical and permeability barriers within the body. All tissues are divided into discrete compartments, each of which is composed of a specific cell type that adheres to similar cell types. Such adhesion triggers the formation of intercellular junctions (i.e., readily definable contact sites on the surfaces of adjacent cells that are adhering to one another), also known as tight junctions, gap junctions and belt desmosomes. The formation of such junctions gives rise to physical and permeability barriers that restrict the free passage of cells and other biological substances from one tissue compartment to another. For example, the blood vessels of all tissues are composed of endothelial cells. In order for components in the blood to enter a given tissue compartment, they must first pass from the lumen of a blood vessel through the barrier formed by the endothelial cells of that vessel. Similarly, in order for substances to enter the body via the gut, the substances must first pass through a barrier formed by the epithelial cells of that tissue. To enter the blood via the skin, both epithelial and endothelial cell layers must be crossed.
Cell adhesion is mediated by specific cell surface adhesion molecules (CAMs). There are many different families of CAMs, including the immunoglobulin, integrin, selectin and cadherin superfamilies, and each cell type expresses a unique combination of these molecules. Cadherins are a rapidly expanding family of calcium-dependent CAMs (Munro et al., In: Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp. 17-34, RG Landes Co.(Austin Tex., 1996). The classical cadherins (abbreviated CADs) are integral membrane glycoproteins that generally promote cell adhesion through homophilic interactions (a CAD on the surface of one cell binds to an identical CAD on the surface of another cell), although CADs also appear to be capable of forming heterotypic complexes with one another under certain circumstances and with lower affinity. Cadherins have been shown to regulate epithelial, endothelial, neural and cancer cell adhesion, with different CADs expressed on different cell types. N (neural)-cadherin is predominantly expressed by neural cells, endothelial cells and a variety of cancer cell types. E (epithelial)-cadherin is predominantly expressed by epithelial cells. Other CADs are P (placental)-cadherin, which is found in human skin and R (retinal)-cadherin. A detailed discussion of the classical cadherins is provided in Munro S B et al., 1996, In: Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp. 17-34 (RG Landes Company, Austin Tex.).
The structures of the CADs are generally similar. As illustrated in FIG. 1, CADs are composed of five extracellular domains (EC1-EC5), a single hydrophobic domain (TM) that transverses the plasma membrane (PM), and two cytoplasmic domains (CP1 and CP2). The calcium binding motifs DXNDN (SEQ ID NO: 1), DXD and LDRE (SEQ ID NO:2) are interspersed throughout the extracellular domains. The first extracellular domain (EC1) contains the classical cadherin cell adhesion recognition (CAR) sequence, HAV (His-Ala-Val), along with flanking sequences on either side of the CAR sequence that may play a role in conferring specificity. Synthetic peptides containing the CAR sequence and antibodies directed against the CAR sequence have been shown to inhibit CAD-dependent processes (Munro et al., supra; Blaschuk et al., J. Mol. Biol. 211:679-82, 1990; Blaschuk et al., Develop. Biol. 139:227-29, 1990; Alexander et al., J. Cell. Physiol. 156:610-18, 1993). However, the determination of the three-dimensional solution and crystal structures of the EC1 domain of classical cadherins (Overduin et al., Science 267:386-389, 1995; Shapiro et al., Nature 374:327-337, 1995) suggest that amino acid residues other than HAV may be directly involved in mediating the interactions between cadherins.
Although cell adhesion is required for certain normal physiological functions, there are situations in which the level of cell adhesion is undesirable. For example, many pathologies (such as autoimmune diseases, cancer and inflammatory diseases) involve abnormal cellular adhesion. Cell adhesion may also play a role in graft rejection. In such circumstances, modulation of cell adhesion may be desirable.
In addition, permeability barriers arising from cell adhesion create difficulties for the delivery of drugs to specific tissues and tumors within the body. For example, skin patches are a convenient tool for administering drugs through the skin. However, the use of skin patches has been limited to small, hydrophobic molecules because of the epithelial and endothelial cell barriers Similarly, endothelial cells render the blood capillaries largely impermeable to drugs, and the blood/brain barrier has hampered the targeting of drugs to the central nervous system. In addition, many solid tumors develop internal barriers that limit the delivery of anti-tumor drugs and antibodies to inner cells.
Attempts to facilitate the passage of drugs across such barriers generally rely on specific receptors or carrier proteins that transport molecules across barriers in vivo. However, such methods are often inefficient, due to low endogenous transport rates or to the poor functioning of a carrier protein with drugs. While improved efficiency has been achieved using a variety of chemical agents that disrupt cell adhesion, such agents are typically associated with undesirable side-effects, may require invasive procedures for administration and may result in irreversible effects.
Accordingly, there is a need in the art for compounds that modulate cell adhesion and improve drug delivery across permeability barriers without such is advantages. The present invention fulfills this need and further provides other elated advantages.
The present invention provides compositions and methods for modulating cadherin-mediated functions. Within certain aspects, the present invention provides cell adhesion modulating agents capable of binding to the cadherin CAR sequence HAV, wherein the agent does not comprise an antibody or antigen-binding fragment thereof.
Within related aspects, the present invention provides cell adhesion modulating agents, comprising: (a) an HAV-BM sequence or peptidomimetic thereof; (b) a polynucleotide encoding an HAV-BM sequence; or (c) an antibody or antigen-binding fragment thereof that specifically binds to an HAV-BM sequence; wherein the agent modulates a cadherin-mediated process. Within certain specific embodiments, the HAV-BM sequence is: (a) Ile/Val-Phe-Aaa-Ile-Baa-Caa-Daa-Ser/Thr-Gly-Eaa-Leu/Met (SEQ ID NO:3), wherein Aaa, Baa, Caa, Daa and Eaa are independently selected from the group consisting of amino acid residues; (b) Trp-Leu-Aaa-Ile-Asp/Asn-Baa-Caa-Daa-Gly-Gln-Ile (SEQ ID NO:4), wherein Aaa, Baa, Caa and Daa are independently selected from the group consisting of amino acid residues; or (c) an analogue of any of the foregoing sequences that retains at least seven consecutive amino acid residues, wherein the ability of the analogue to modulate a cadherin-mediated process is not diminished. For example, a cell adhesion modulating agent may comprise an HAV-BM sequence is selected from the group consisting of: IFIINPISGQL (SEQ ID NO:5), IFILNPISGQL (SEQ ID NO:6), VFAVEKETGWL (SEQ ID NO:7), VFSINSMSGRM (SEQ ID NO:8), VFIIERETGWL (SEQ ID NO:9), VFTIEKESGWL (SEQ ID NO:10), VFNIDSMSGRM (SEQ ID NO:11), WLKIDSVNGQI (SEQ ID NO:12), WLKIDPVNGQI (SEQ ID NO:13), WLAMDPDSGQV (SEQ ID NO:14), WLHINATNGQI (SEQ ID NO:15), WLEINPDTGAI (SEQ ID NO:16), WLAVDPDSGQI (SEQ ID NO:17), WLEINPETGAI (SEQ ID NO:18), WLHINTSNGQI (SEQ ID NO:19), NLKIDPVNGQI (SEQ ID NO:20), LKIDPVNGQI (SEQ ID NO:21) and analogues of the foregoing sequences that retain at least seven consecutive residues (e.g., INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQ ID NO:24) or KIDPVNGQ (SEQ ID NO:25)), wherein the ability of the analogue to modulate a cadherin-mediated process is not diminished. Alternatively, a modulating agent may comprise an HAV-BM sequence that comprises at least five consecutive residues of a peptide selected from the group consisting of INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), NLKIDPVNGQI (SEQ ID NO:20) and WLKIDPVNGQI (SEQ ID NO: 13). For example, the agent may comprise a sequence selected from the group consisting of PISGQ (SEQ ID NO:26), PVNGQ (SEQ ID NO:27), PVSGR (SEQ ID NO:28), IDPVN (SEQ ID NO:29), INPIS (SEQ ID NO:30) and KIDPV (SEQ ID NO:3 1). Within such modulating agents, an HAV-BM sequence may be present within a linear peptide or a cyclic peptide. Certain modulating agents comprise a cyclic peptide having one of the formulas: 
wherein X1, and X2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds, and wherein X1 and X2 independently range in size from 0 to 10 residues, such that the sum of residues contained within X1 and X2 ranges from 1 to 12; wherein Y1 and Y2 are independently selected from the group consisting of amino acid residues, and wherein a covalent bond is formed between residues Y1 and Y2; and wherein Z1 and Z2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds. Such cyclic peptides may contain modifications. For example, Y1 may comprise an N-acetyl group and/or Y2 may comprise a C-terminal amide group. Cyclization may be achieved in any of a variety of ways, such as covalent linkage of Y1 and Y2 via a disulfide, amide or thioether bond.
Within certain embodiments, modulating agents as described above may be linked to one or more of a drug, a solid support, a detectable marker or a targeting agent.
Within other embodiments, a modulating agents as described above may further comprise one or more of: (a) a cell adhesion recognition sequence other than an HAV-BM sequence, wherein the cell adhesion recognition sequence is separated from any HAV-BM sequence(s) by a linker; and/or (b) an antibody or antigen-binding fragment thereof that specifically binds to a cell adhesion recognition sequence other than an HAV-BM sequence. For example, the adhesion molecule may be selected from the group consisting of cadherins, integrins, occludin, N-CAM, desmogleins. desmocollins, fibronectin, laminin and other extracellular matrix proteins.
Within further aspects, the present invention provides pharmaceutical compositions comprising a cell adhesion modulating agent as described above, in combination with a pharmaceutically acceptable carrier. Such compositions may further comprise one or more of a drug and/or a modulator of cell adhesion, wherein the modulator comprises one or more of: (a) a peptide comprising a cell adhesion recognition sequence other than an HAV-BM sequence; and/or (b) an antibody or antigen-binding fragment thereof that specifically binds to a cell adhesion recognition sequence other than an HAV-BM sequence. For example, the adhesion molecule may be selected from the group consisting of cadherins, integrins, occludin, N-CAM, desmogleins, desmocollins, fibronectin, laminin and other extracellular matrix proteins.
The present invention further provides, within other aspects, methods for modulating a cadherin-mediated function, comprising contacting a cadherin-expressing cell with a cell adhesion modulating agent as described above. Cadherin-mediated functions include cell adhesion, neurite outgrowth, Schwann cell migration and synaptic stability. Cadherin-expressing cells include epithelial cells, endothelial cells, neural cells, tumor cells and lymphocytes. Within such aspects, the cell adhesion modulating agent may inhibit or enhance a cadherin-mediated function.
Within other aspects, the present invention provides methods for reducing unwanted cellular adhesion in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent inhibits cadherin-mediated cell adhesion. The cell may be selected from the group consisting of epithelial cells, endothelial cells, neural cells, tumor cells and lymphocytes.
The present invention further provides, within other aspects, methods for enhancing the delivery of a drug through the skin of a mammal, comprising contacting epithelial cells of a mammal with a drug and a modulating agent as described above, wherein the step of contacting is performed under conditions and for a time sufficient to allow passage of the drug across the epithelial cells, and wherein the modulating agent inhibits cadherin-mediated cell adhesion. The modulating agent may, but need not, be linked to the drug and may, within certain embodiments, pass into the blood stream of the mammal. The step of contacting may be performed via a skin patch comprising the modulating agent and the drug.
Within further aspects, methods are provided for enhancing the delivery of a drug to a tumor in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent inhibits cadherin-mediated cell adhesion. Suitable tumors include, but are not limited to, bladder tumors, ovarian tumors and melanomas, and the modulating agent may be administered to the tumor or systemically.
Within other aspects, the present invention provides methods for treating cancer and/or inhibiting metastasis in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent inhibits cadherin-mediated cell adhesion. The mammal may be afflicted with a cancer such as a carcinoma, leukemia or melanoma, and the modulating agent may be administered to the tumor or systemically.
The present invention further provides, within other aspects, methods for inducing apoptosis in a cadherin-expressing cell, comprising contacting a cadherin-expressing cell with a modulating agent as described above, wherein the modulating agent inhibits cadherin-mediated cell adhesion.
Within other aspects, methods are provided for inhibiting angiogenesis in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent inhibits cadherin-mediated cell adhesion.
The present invention further provides, within other aspects, methods for enhancing drug delivery to the central nervous system of a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent inhibits cadherin-mediated cell adhesion.
Within further aspects, the present invention provides methods for facilitating wound healing in a mammal, comprising contacting a wound in a mammal with a modulating agent as described above, wherein the modulating agent enhances cadherin-mediated cell adhesion.
Methods are also provided, within other aspects, for enhancing adhesion of foreign tissue implanted within a mammal, comprising contacting a site of implantation of foreign tissue in a mammal with a modulating agent as described above, wherein the modulating agent enhances cadherin-mediated cell adhesion. Such foreign tissue may be a skin graft or organ implant. Within certain embodiments, the modulating agent is linked to a support material.
The present invention further provides, in other aspects, methods for enhancing and/or directing neurite outgrowth, comprising contacting a neuron with a modulating agent as described above, wherein the modulating agent enhances cadherin-mediated cell adhesion.
Within other aspects, the present invention provides methods for treating spinal cord injuries in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent enhances cadherin-mediated cell adhesion.
Methods are also provided, within further aspects, for treating a demyelinating neurological disease such as multiple sclerosis in a mammal, comprising administering to a mammal a modulating agent as described above. Within certain embodiments, the modulating agent is administered by implantation with Schwann cells, oligodendrocyte progenitor cells and/or oligodendrocytes.
Within further aspects, methods are provided for modulating the immune system of a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent inhibits cadherin-mediated cell adhesion.
Within other aspects, the present invention provides methods for preventing pregnancy in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent inhibits cadherin-mediated cell adhesion.
Methods are further provided for increasing vasopermeability in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent inhibits cadherin-mediated cell adhesion.
Within further aspects, the present invention provides methods for inhibiting synaptic stability in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent inhibits cadherin-mediated cell adhesion.
The present invention further provides methods for detecting the presence of cadherin-expressing cells in a sample, comprising: (a) contacting a sample with an antibody or antigen-binding fragment thereof that binds to an HAV-BM sequence under conditions and for a time sufficient to allow formation of an antibody-cadherin complex; and (b) detecting the level of antibody-cadherin complex, and therefrom detecting the presence of cadherin expressing cells in a sample. The antibody may be linked to a support material or a detectable marker such as a fluorescent marker. In certain embodiments, the step of detecting is performed using fluorescence activated cell sorting.
The present invention also provides, within further aspects, kits for enhancing transdermal drug delivery, comprising: (a) a skin patch; and (b) a modulating agent as described above. The skin patch may be impregnated with the modulating agent, and the kit may further comprise a drug.
Kits for detecting the presence of cadherin-expressing cells in a sample are also provided. Such kits may comprise: (a) an antibody or antigen-binding fragment thereof that specifically binds to an HAV-BM sequence; and (b) a detection reagent.
Within other aspects, the present invention provides methods for identifying a compound capable of modulating cadherin-mediated cell adhesion, comprising: (a) contacting an antibody or antigen-binding fragment thereof that specifically binds to an HAV-BM sequence with a test compound; and (b) detecting the level of antibody or fragment that binds to the test compound, and therefrom identifying a compound capable of modulating cadherin-mediated cell adhesion.
Methods are also provided, within other aspects, for facilitating blood sampling in a mammal, comprising contacting epithelial cells of a mammal with a cell adhesion modulating agent as described above, wherein the modulating agent inhibits cadherin-mediated cell adhesion, and wherein the step of contacting is performed under conditions and for a time sufficient to allow passage of one or more blood components across the epithelial cells. The step of contacting may be performed via a skin patch comprising the modulating agent, and the skin patch may further comprise a reagent for detecting a blood component of interest. Within certain embodiments, the epithelial cells are skin cells or gum cells.
Within related aspects, the present invention provides kits for sampling blood via the skin or gum of a mammal, comprising: (a) a skin patch; (b) a cell adhesion modulating agent comprising a cyclic peptide that comprises a cadherin CAR sequence; and (c) a reagent for detecting a blood component of interest. The skin patch may be impregnated with the cell adhesion modulating agent.
Within other aspects, the present invention provides methods for screening for a compound that interacts with an HAV-BM sequence, comprising the steps of: (a) contacting a candidate compound with an HAV-BM sequence; and (b) evaluating the ability of the candidate compound to bind to the HAV-BM sequence, and therefrom determining whether the candidate compound interacts with an HAV-BM sequence. Within certain embodiments, the ability of the candidate compound to bind to the HAV-BM sequence is evaluated using an affinity column or a Western blot analysis. Within certain embodiments, the candidate compound is encoded by a polynucleotide in an expression library, or the candidate compound is a cellular protein, and step (b) is performed using whole cells.
These and other aspects of the invention will become evident upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each were individually noted for incorporation.